SARS-CoV-2 impairs male fertility by targeting semen quality and testosterone level: A systematic review and meta-analysis

Background Since the discovery of COVID-19 in December 2019, the novel virus has spread globally causing significant medical and socio-economic burden. Although the pandemic has been curtailed, the virus and its attendant complication live on. A major global concern is its adverse impact on male fertility. Aim This study was aimed to give an up to date and robust data regarding the effect of COVID-19 on semen variables and male reproductive hormones. Materials and methods Literature search was performed according to the recommendations of PRISMA. Out of the 852 studies collected, only 40 were eligible for inclusion in assessing the effect SARS-CoV-2 exerts on semen quality and androgens. More so, a SWOT analysis was conducted. Results The present study demonstrated that SARS-CoV-2 significantly reduced ejaculate volume, sperm count, concentration, viability, normal morphology, and total and progressive motility. Furthermore, SARS-CoV-2 led to a reduction in circulating testosterone level, but a rise in oestrogen, prolactin, and luteinizing hormone levels. These findings were associated with a decline in testosterone/luteinizing hormone ratio. Conclusions The current study provides compelling evidence that SARS-CoV-2 may lower male fertility by reducing semen quality through a hormone-dependent mechanism; reduction in testosterone level and increase in oestrogen and prolactin levels.

As of May 2023, over 766 million COVID-19 cases, with about 7 million mortalities were reported [9].Studies have revealed that COVID-19 mainly affects both male and female respiratory systems [4,8].Studies have also demonstrated that the virus causes damage to multiple organs, including the kidney, heart, liver, brain [10,12], and testes [2,4,6,8,13].In addition, there is proof that SARS-CoV-1 exerts a more severe impact on males than females [6,[14][15][16][17].Also, orchitis has been reported in males recovering from the SARS virus [3,18].Despite this, findings on the adverse effect of this deadly virus on the male reproductive system are limited and contentious.In a systematic review and meta-analysis by Corona et al. [21], SARS-CoV-2 infection was linked with low semen quality and serum testosterone level.This is in agreement with earlier systematic review and meta-analysis by Tiwari et al. [22].The study however had some frailties-first, the random-effect model was used irrespective of the level of diversity, which might affect the findings of the meta-analysis.Also, no sensitivity analyses were performed to rule out the influence of diversity.Finally, the authors failed to apply the finding of the quality of the appraised studies to their analysis.
Therefore, the aim of this study is oriented towards providing an overhauling meta-analysis on the consequence of COVID-19 on male fertility.This review gives an insight into how COVID-19 impact semen quality and male reproductive hormones to modulate male fertility.So far as we are aware, this research pioneers the evaluation of the impact of COVID-19 by comparing between infected and non-infected subjects, before and after treatment in infected patients, and infected and pre-COVID state in the same patients.Hence, the present study evinces a robust review and analysis of the influence of SARS-CoV-2 on male fertility.

Protocol and eligibility criteria for inclusion
This study was registered on Prospero (CRD42024533906).This study was conducted on published works that evaluated the influence of SARS-CoV-2 on male fertility.The study adopted the "Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA)" strategy, which is provided as Fig 1.This study adopted the Population, Exposure, Comparator/Comparison, and Outcomes (PECO) model.All studies published until October 2023 that were eligible based on set criteria, were collected.The studied populations were male in their reproductive ages, who had an exposure to SARS-CoV-2 and developed COVID 19.The studies were either retrospective or prospective among COVID 19-infected patients with age-matched control who were COVID 19-negative.In cases where there were no COVID 19-negative control groups, outcomes before and after the treatment of COVID 19 or at pre-COVID and COVID 19-infected states should be presented.The outcome measured were conventional semen parameters viz.ejaculate volume, sperm count, concentration, viability, normal morphology, total and progressive motility, and seminal fluid leukocyte level, and male reproductive hormones namely testosterone (T), oestrogen, prolactin, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels.T/LH and FSH/LH were also measured.
Exclusion criteria included absence of a comparator as control, studies in females, in vitro studies, commentaries, review articles, letters to editor, editorials, preprint, conference abstracts, retracted papers, and degree thesis.No language or country restriction was applied.

Data collection, assessment of quality of eligible studies, and meta-analysis
The eligible studies were appraised for quality and data collected by AVJ, APJ, and .ATM. Disputes were resolved by ARE.Data gathered from the appropriate studies include the last name of the principal investigator, publication date, country of study origin, study design, method of COVID 19 diagnosis, sample size and ages of patients, duration of infection, and measured outcomes of interest.The outcomes of interest were pull out as mean and standard deviation.When the variables were presented in other forms, the mean and standard deviation were derived from the provided data.In cases where the outcomes were reported in Figs, they were converted to values using Web Plot Digitizer.
The quality of evidence in the eligible papers was evaluated using the ErasmusAGE quality score for systematic reviews, which assigns a number between 0 and 2 to five domains [23].Furthermore, the "Office of Health Assessment and Translation (OHAT)" methodology was used to evaluate the risk of bias (RoB) [24].Using the "Grading of Recommendations Assessment, Development and Evaluation (GRADE) Working Group" standards as a guide, the "OHAT approach for systematic review and evidence integration for literature-based health assessment was used to assess the certainty of the evidence" [25,26].
Review Manager (version 5.4.1) was used to conduct the quantitative meta-analyses.From the eligible studies, the standardized mean difference (SMD) at 95% confidence intervals (CIs) was calculated.A random-effect model was used when P-value < 0.1 or I2 > 50% which indicates the existence of significant variety; otherwise, a fixed-effect model was utilized.To assess the possible sources of diversity, sensitivity analysis was conducted by excluding the studies with the largest weight, high RoB (< 4), low quality of evidence (< 5) and low certainty of evidence.Also, the generated funnel's plots were visually assessed for publication bias.

The selection of studies and the attributes of the relevant studies
Out of the 852 publications screened, only 50 were potentially eligible for evaluation.Finally, 40 studies  were deemed eligible for inclusion in this study (Fig 1).The eligible papers were published between 2020 and 2023, and they were from China (7), Germany (1), India (1), Indonesia (1), Iran (6), Iraq (2), Italy (5), Jordan (2), Russia (1), Turkey (12), UK (1), and USA (1).The data collected included the surname of the year of publication, principal investigator, country of study origin, study design, method of diagnosing COVID-19, studied population size, participants'/patients' age range, duration of infection, outcomes measured (Table 1).

Assessment of the quality of evidence, RoB, and certainty of evidence
A larger part of the studies had good quality of evidence, except 7 of them [27,31,40,48,50,56,64] that had low quality of evidence (<5) (Table 2).Also, the included studies had moderate (4/9-6/9) to low (>6/9) RoB (Table 3).In addition, the certainty of evidence in the included studies were moderate to high, except in 3 studies [29,48,56] with low certainty of evidence (Table 4).

Discussion
Although the achievement of clinical pregnancy and live birth is the true test of infertility, conventional semen analysis remains the cornerstone of the diagnosis and management of male infertility [67].Evaluation of male sex hormones is also a useful tool in the management of male infertility.Our present data revealed that SARS-CoV-2 caused reductions in ejaculate volume, sperm count, concentration, viability, normal morphology, and total and progressive motility.These findings were associated with SARS-CoV-2-induced decline in serum testosterone level, and increase in oestrogen, prolactin, LH, and testosterone/LH levels.These data convincingly demonstrate that SARS-CoV-2 may impede fertility in males by engendering a nadir of semen quality and distorting male reproductive hormone milieu.
The present findings corroborate and form an extension of the previous findings of the meta-analysis of Corona et al. [21], Tiwari et al. [22], and Xie et al [68].Our present findings provide an update and robust data demonstrating the detrimental sequelae of SARS-CoV-2 on semen quality and male sex hormones.These data also augment the evidence available in the scientific literature that support the grievous consequence which SARS-CoV-2 impacts on male reproductive function.
It is plausible to infer that SARAS-CoV-2 may impair male fertility through multiple pathways.The expression of SARS-CoV-2 virus in the semen of infected patients [69][70][71] suggests that the virus may exert a local effect on the sperm cells.SARS-CoV-2 virus promotes oxidative stress evinced by heightened reactive oxygen species (ROS) generation, malondialdehyde (MDA) level and decline in total antioxidant capacity (TAC) in the semen fluid of infected patients [38].Since the sperm cells are rich in polyunsaturated fatty acids that make them highly susceptible to ROS attack, SARS-CoV-2-induced ROS generation in the spermatozoa may cause oxidative sperm damage, leading to reduced sperm count, viability, motility, concentration, and normal morphology.
In addition, studies have shown that SARS-CoV-2 positively modulates cytokines 30 through extracellular-regulated protein kinase (ERK) and p38 mitogen-activated protein kinases (MAPK) activation [3,4,72], thus activating a cascade of immune responses, which lead to a hyper-inflammatory state that compromise the blood-testis-barrier [3,73,74] and increase the susceptibility of the testis and germ cells to SARS-CoV-2-driven ROS attack.This may explain the reduced semen quality and testosterone levels observed in SARS-CoV-2 positive patient.Since LH and FSH levels were not reduced in association with reduced testosterone, it is  Beyond semen quality, SARS-CoV-2 infection may also impact on the success of testicular sperm extraction, hence on the outcome of assisted reproductive techniques (ART).Testosterone/LH is a known predictor of sperm concentration and successful sperm retrieval [75,76]; therefore, the reduced testosterone/LH level in SARS-CoV-infected patients explains the reduced sperm concentration found in the patients and also reveals a likelihood of reduced success rate of sperm retrieval in them.This implies that SARS-CoV-2 may lower the rate of spontaneous conception as well as reduce the success of ARTs.Since testosterone/LH is also a predictor of Leydig cell function [76,77], it is also credible to infer that SARS-CoV-2 impairs Leydig cell function.This may the reduced testosterone found in SARS-CoV-2 positive men.
It is imperative to note that the duration of the infection and time between infection and semen collection might have an effect on the study outcomes.Findings of Koc ¸and Keseroğlu [48], and Temiz et al. [63] that performed semen analysis after 5 and 4 days of infection respectively showed insignificant changes for most of the sperm variables and testosterone level.It is also worth mentioning that most of the eligible studies were published between 2020 and 2022, indicating that they were likely before the introduction of COVID-19 vaccines and also before the infection by the most recent and less dangerous variants of COVID-19; hence, the impact of the virus may differ.It is likely that COVID-19 vaccination confers protection against sperm-endocrine aberrations induced by the novel virus.More so, the less virulent variants of COVID-19 may exert less adverse effect on the sperm-endocrine system than the virulent variant.Just like other systematic viral infections, SARS-CoV-2 impairs male fertility possibly by upregulating pro-inflammatory cytokines and promoting hyper-inflammation and oxidative stress or direct sperm-endocrine alterations [3].The peculiarity of SARS-CoV-2 hinges around its novelty.
Despite the fascinating and convincing findings of this study, there are some limitations.First, the effect of SARS-CoV-2 on live-birth rate is not presented, which limits our conclusion on the effect of the viral diseases on male fertility.Also, there were remarkable risk of publication bias in many of the studies.More so, the significant diversity in most of the studies is a major concern, although this was controlled by a sensitivity analysis.Lastly, studies exploring the actual mechanisms on SARS-CoV-2 on semen quality and male sex hormones are lacking and most studies were speculative.Nonetheless, the present meta-analysis provides an update and a robust data delineating the consequences of SARS-CoV-2 on conventional semen In conclusion, this study demonstrates that SARS-CoV-2 may diminish fertility in male by reducing semen quality viz.ejaculate volume, sperm count, concentration, viability, motility, and normal morphology through a hormone-dependent mechanism (reduction in testosterone level and increase in oestrogen and prolactin levels).It is also likely that the induction of oxidative stress and inflammatory injury play significant roles.More well-designed studies which accommodate larger sample size should be conducted to validate these findings, evaluate the long term effect of SARS-CoV-2 on sperm function and testosterone concentration, establish the associated mechanisms, and address the weaknesses highlighted are recommended.